The use of high gradient magnetic fields for the separation of particles is commonplace in the fields of biology, biotechnology, and other bio-medical fields. Target particles, comprising entities such as proteins and the like, may be separated from a solution by a technique known as magnetic separation.
In general, magnetic separation of specifically sought after biological entities involves coating small magnetically susceptible paramagnetic, super paramagnetic, or ferromagnetic, materials with a chemical-specific substance (e.g., a ligand that is known to chemically bond with target entities). These coated materials are referred to hereinafter as beads. The beads are introduced into a well containing a hydrous solution of the sought after, or target, entities, and unwanted biological material. Cells, proteins, nucleic acid sequences and the like are examples of target entities. The target entities chemically bond to the coating of the beads. Magnets are placed near the well to apply magnetic fields in the well and the solution. Although it is the presence of substances and other coatings that ultimately interact with the target entities, it is the characteristics of the magnetic field that is applied and physical characteristics of the beads that determine the separation time and the uniformity of the profile of the separated beads. A uniform bead separation profile is desirable, such as a profile in which the beads uniformly distribute about the base of each well to produce a “flat” profile, or in which the beads pull to the sides of the wells equally at every location.
The beads, including the target entities chemically bonded to the coating of the beads, are attracted to the magnets. The magnetic configuration corresponds well with the analytical equation which states that the resultant force on the beads (F) is proportional to the magnitude of the magnetic field (B) multiplied by its gradient (∇B).F∝B·∇B  (1)
For the case of bead separation within a multi-well tray, the hydrous solutions in each well are physically divided. Consequently, to obtain a “uniform” distribution of beads (assuming that each well is using the same size beads, density of beads, and volume of solution) along the entire base of the tray, the above two components (B and ∇B) of the force equation (1) are equal throughout the active volume of the tray, but not necessarily equal to each other. This can be accomplished by shaping a magnet to provide a uniform magnetic field and gradient parallel to the base of each well within the tray. FIG. 1 represents one such configuration.
In the case of positive separation, that is, where the sought after entities are attracted to the beads, once the beads have been collected at the desired location, the well is rinsed, removing the solution and unwanted particles. The collected beads with the target entities chemically bonded to the coating of the beads remain in the well as long as the magnetic fields are applied.
Once the well has been rinsed, a “clean” solution, without unwanted particles, is introduced into the well. A chemical is mixed with the “clean” solution to break the chemical bonds between the target entities and the coating of the beads, resulting in a well with isolated target entities. Additionally, the beads may be removed by disabling/removing the magnetic fields from the well.
In the case of negative separation, that is, where the unwanted entities are attracted to the beads and the sought after entities removed, once the beads have been collected at the desired location, the well is rinsed, removing the solution and sought after particles. The collected beads with the unwanted entities chemically bonded to the coating of the beads remain in the well as long as the magnetic fields are applied.
Molecular biological magnetic separation is well known, and until relatively recently, this process was performed using large tubes of fluids (e.g., 15–50 ml tubes) and beads. Recent molecular magnetic separation techniques typically involve the use of 96-well micro-plates, that is, a tray having 96 wells, arranged in an 8×12 matrix, with each well capable of holding 250–500 micro-liters (μl) of liquid. In another embodiment of the invention, each well may be capable of holding more than 250–500 micro-liters of liquid. A variety of placement methods for magnets to apply the desired magnetic fields may be employed on these micro-plates. One method is to place small magnets, having predetermined magnetic fields, between micro-plate receiving orifices, so that the beads collect along the walls of the wells. Another method is to place an apparatus with magnetic pins into the wells with the beads collecting on the pins. Another method is to have a base for a micro-plate with cylindrical magnets positioned for insertion from the base of the micro-plate into the spaces between the wells of the micro-plate with the beads collecting along the walls of the wells.
As molecular magnetic separation techniques advance, the number of wells increase. In high throughput applications, typically involving automated systems, 384-well to 1536-well micro-plates are utilized to increase capacity and throughput. In such systems, each 384-well micro-plate is arranged as 16×24 wells, while each 1536-well micro-plate is arranged as 32×48 wells, effectively increasing the throughput of the conventional 96-well micro-plate by 4 and 16 times respectively.
As the number of wells increase, the spaces between the individual wells in micro-plates decrease, in some cases, to the point where there is no space between the wells, making the placement of magnets between rows of wells impracticable. However, magnets are still required to separate the target particles from the solution contained in micro-plates.